We are investigating methods for improving the efficiency of gene targeting so that it can be a useful method for human gene therapy. Gene targeting is a recombinational event that produces exchange between DNA introduced into a cell and its homologous chromosomal target. Homologous recombination involves two major steps: pairing of DNA strands at the region of homology, and DNA strand exchange. Homologous recombination allows precise targeted insertion of genetic information, gene expression in appropriate tissues at appropriate levels, correction of mutations including dominant mutations, and gene inactivation. It is known from experiments in bacteria and yeast that homologous recombina-tion events are mediated by general recombinases. General recombination proteins function by recognizing and pairing DNA sequences on the basis of shared homology. The best characterized general recombinase is the bacterial recA protein. RecA alone catalyzes the pairing and strand exchange steps during homologous recombination. Initially, we performed gene targeting experiments in mouse embryonic stem (ES) cells in the presence of recA. We had created mouse ES cells that stably express recA, with and without a nuclear localization signal, under inducible promoters. In order to determine the efficiency of homologous recombination in the various genetically engineered mouse ES cells, we designed a method to perform hundreds of gene targeting experiments efficiently. The method that we created significantly increases the number of mouse ES cells that are targeted by homologous recombination. This method is not dependent on the presence of recA which suggests that mouse ES cells contain sufficient quantities of general recombinases in order to perform gene targeting efficiently. Our procedure produces a 1,000-1,000,000 fold increase in the currently reported gene targeting frequencies in mouse ES cells. Until now, the efficiency of homologous recombination has been too low for use in human gene therapy. Gene targeting can now be used for human gene correction or gene inactivation. This method could be a significant contribution to the production of transgenic animals.